PSVIII-12 Comparative characterization of intestinal alkaline phosphatase kinetics in young piglets and human Caco-2 cells

摘要

This study reports the kinetics of young porcine jejunal alkaline phosphatase (IAP) towards the dephosphorylation of ATP, lipopolysaccharides (LPS) and p-nitrophenyl phosphate (pNPP) at the physiological conditions (pH = 7.4; and at 37 °C) in comparison with the IAP from human Caco-2 cells. The 10-day suckling young porcine jejunal IAP displayed the Km values of 1.26±0.50 mM, 1.35±0.64 mg/mL and 0.290±0.072 mM for the hydrolyses of ATP, LPS and pNPP, respectively; while the respective Km values were 0.030±0.007 mM, 0.66±0.22 mg/mL and 0.033±0.006 mM for the IAP in the Caco-2 cells towards the same set of substrates. In comparison, the Km values of the young porcine jejunal IAP were 2–40 times higher than those of IAP from the human Caco-2 cells. In addition, Pearson correlation analyses showed tight positive correlations (P < 0.001) between IAP activities towards pNPP, ATP and LPS in the piglet and the Caco-2 cells, suggesting that the IAP activity towards pNPP could be used to predict the IAP activities on the physiological substrates of ATP and LPS. Lastly, four AP genes were identified in the genome of Sus scrofa. Three of these porcine AP genes are annotated as intestinal-type alkaline phosphatase (IAP) genes and clustered at the distal end of chromosome 15, namely IAPX1, IAPX2, and IAPX3. The genomic context of APs in the pig genome is highly similar to those in the human genome. We predict that pig IAPX3 ({"type":"entrez-protein","attrs":{"text":"XP_003133777.1","term_id":"311273235","term_text":"XP_003133777.1"}}XP_003133777.1) is likely an IAP gene for pigs. Further comparisons in post-translational modification and protein 3-D structure modelling were performed, indicating that the observed differences in kinetic affinity between the young porcine jejunal IAP and the human Caco-2 cell IAP in hydrolyzing ATP, LPS and pNPP might relate to their differences in the coding sequences of the IAP protein, and/or the IAP protein N-glycosylation.